The Ubiquitin System
Several biological processes are controlled by the ubiquitination of cellular protein. Cellular processes that are affected by ubiquitin modification include the regulation of gene expression, regulation of the cell cycle and cell division, cellular housekeeping, cell-specific metabolic pathways, disposal of mutated or post-translationally damaged proteins, the cellular stress response, modification of cell surface receptors, DNA repair, import of proteins into mitochondria, uptake of precursors into neurons, biogenesis of mitochondria, ribosomes, and peroxisomes, apoptosis, and growth factor-mediated signal transduction.
For some protein substrates ubiquitination leads to protein degradation by the 26S proteasomal complex. A wide variety of protein substrates are degraded by the 26S proteasomal complex following ubiquitination of the substrate. Degradation of a protein by the ubiquitin system involves two steps. The first involves the covalent attachment of multiple ubiquitin molecules to the substrate protein. The second involves degradation of the ubiquitinated protein by the 26S proteasome. In some cases, degradation of the ubiquitinated protein can occur by means of the lysosomal pathway.
The 26S proteasome comprises a 20S core catalytic complex which is flanked by two 19S regulatory complexes. The 26S complex recognizes ubiquitinated proteins. Substrate recognition by the 26S proteasome, however, may be mediated by the interaction of specific subunits of the 19S complex with the ubiquitin chain. The ubiquitinated protein is degraded by specific and energy-dependent proteases into free amino acids and free and reutilizable ubiquitin.
The 19S regulatory complex consists of many subunits that can be classified into ATPases and non-ATPases. This complex is thought to act in recognition, unfolding, and translocation of the substrates into the 20S proteasome for proteolysis. The regulatory complex contains isopeptidases capable of deubiquitinating substrates (Spataro et al. (1998) British Journal of Cancer 77:448-455).
The ubiquitin proteasome pathway functions to degrade abnormal proteins, short-lived normal proteins, long-lived normal proteins, and proteins of the endoplasmic recticulum. Important regulatory proteins rapidly inactivated by proteolysis include c-JUN, c-FOS, and p53 (Lecker et al. (1999) Journal of Nutrition 129:227S-237S). Conditions that stimulate protein degradation by the ubiquitin proteasome pathway include eating disorders, renal tubular defects, diabetes, uremia, neuromuscular disease, immobilization, burn injuries, sepsis, cancer, cachexia, hyperadrenocortisolism and hyperthyroidism.
Cellular proteins degraded by the ubiquitin system include cell cycle regulators, including mitotic cyclins, G1 cyclins, CDK inhibitors, anaphase inhibitors, transcription factors, tumor suppressors, and oncoproteins such as NF-.kappa.B and I.kappa.B.alpha., p53, JUN, .beta.-catenin, E2F-1, and membrane proteins such as Ste2p, GH receptor, T-cell receptor, platelet-derived growth factor, lymphocyte homing receptor, MET tyrosine kinase receptor, hepatocyte growth factor-scatter factor, connexin 43, the high affinity IgE receptor, the prolactin receptor, and the EGF receptor (Hershko et al. (1998) Annual Review of Biochemistry 67:425-479).
Ubiquitination does not only result in proteolytic degradation. For some protein substrates, ubiquitination is a reversible post-translational modification that can regulate cellular targeting and enzymatic activity. This includes targeting to the vacuole, activation of enzyme activity, such as Ik.beta. kinase activation, and activation of cytokine receptor-mediated signal transduction (D'Andrea et al. (1998) Critical Reviews In Biochemistry and Molecular Biology 33:337-352). The T-cell receptor undergoes ubiquitination in response to receptor engagement. Platelet derived growth factor undergoes multiple ubiquitination following ligand binding. Soluble steel factor has been shown to stimulate rapid polyubiquitination of the c-KIT receptor.
It has been shown that protein degradation accounts for regulation of proteins such as cyclins, cyclin-dependent kinase inhibitors, p53, c-JUN and c-FOS (Spitaro et al. above). The ubiquitin system has also shown to be involved in antigen presentation. The 26S proteasome is responsible for processing MHC-restricted class I antigens (Spitaro et al. above).
The ubiquitin system has been implicated in various diseases. One group includes pathology that results from loss of function, a mutation in an enzyme or substrate that leads to stabilization of the protein and consequent build up of a protein to abnormally high levels. The second involves pathologies that result from a gain of function that produces increased protein degradation.
The ubiquitin system has been implicated in various malignancies. In cervical carcinoma, low levels of p53 have been found. This protein is targeted for degradation by HPV E6-associated protein. Removal of the suppressor by this oncoprotein may be a mechanism utilized by the virus to transform cells. Other results have shown that c-JUN, but not the transforming counterpart, v-JUN, is ubiquitinated and subsequently degraded. Other studies show that low levels of p27, a cell division kinase inhibitor whose degradation is necessary for proper cell cycle progression, is correlated with colorectal, and breast carcinomas. The low level of this enzyme is due to activation of the ubiquitin system.
Human genetic diseases involving aberrant proteolysis have been reviewed (Kato (1999) Human Mutation 13:87-98). Cystic fibrosis has been correlated with the ubiquitin system. The cystic fibrosis transmembrane regulator in cystic fibrosis patients is almost completely degraded by the ubiquitin system so that an abnormally low amount of the wild type protein is found on the cell surface. In Angelman's syndrome, one of the enzymes involved in ubiquitination (E3) is affected. In Liddle syndrome, the E3 enzyme is also affected.
The ubiquitin system can also affect the immune and inflammatory response. The persistence of EBNA-1 contributes to some virus related pathologies. A sequence on this protein was found to inhibit degradation by the ubiquitin system. This inhibited processing and subsequent presentation of viral epitopes by SMC protein.
The ubiquitin system has also been implicated in neurodegenerative diseases. Ubiquitin immunohistochemistry has shown enrichment of ubiquitin conjugates in senile plaques, lysosomes, endosomes, and a variety of inclusion bodies and degenerative fibers in many neurodegenerative diseases, such as Alzheimer's, Parkinson's and Lewy body diseases, amyotrophic lateral sclerosis, and Creutzfeld-Jakob disease. Further, in Huntington disease and spinocerebellar ataxias, the proteins encoded by the affected genes aggregate in ubiquitin- and proteasome-positive intranuclear inclusion bodies.
The ubiquitin system has been associated with muscle wasting (Mitch et al. (1999) American Journal of Physiology 276: C1132-C1138 and Lecker et al. above) and muscle-wasting diseases and in such pathological states as fasting, starvation, sepsis, and denervation, all of which result from accelerated ubiquitin-mediated proteolysis (see Ciechanover, EMBO Journal 17:7151-7160 (1998)).
The ubiquitin system is also involved in development. The involvement in human brain development is indicated by the fact that a mutation in an E3 enzyme is implicated as the cause of Angelman's syndrome, a disorder characterized by mental retardation, seizures, and abnormal gait (Hershko et al. above).
The ubiquitin system is also associated with apoptosis. Ubiquitin-proteasome-mediated proteolysis is reported to play an important role in apoptosis of nerve growth factor-deprived neurons (Sadoul et al. (1996) EMBO Journal 15:3845-3852). One of the first genes shown to be involved in programmed cell death is the polyubiquitin gene that is regulated during metamorphosis of Manduca sexta. Radiation-induced apoptosis in human lymphocytes has been shown to be accompanied by increased ubiquitin mRNA and ubiquitinylated nuclear proteins. Further, drugs that interfere with proteasome function, such as lactacystin, prevent radiation-induced cell death of thymocytes (Hershko et al. above).
Deubiquitinating Enzymes
Deubiquitinating enzymes are cysteine proteases that specifically cleave ubiquitin conjugates at the ubiquitin carboxy terminus. These enzymes are responsible for processing linear polyubiquitin chains to generate free ubiquitin from precursor fusion proteins. They also affect pools of free ubiquitin by recycling branched chain ubiquitin. These enzymes also remove ubiquitin from ubiquitin- and polyubiquitin-conjugated target protein, thereby regulating localization or activity of the target. Further, these enzymes can remove ubiquitin from a ubiquitinated tagged protein and thereby rescue the protein from degradation by the 26S proteasome. The end result of each of these activities, is to affect the level of free intracellular ubiquitin (D'Andrea et al., above) and the level of specific proteins.
Ubiquitin is synthesized in a variety of functionally-distinct forms. One of these is a linear head-to-tail polyubiquitin precursor. Release of the free molecules involves specific enzymatic cleavage between the fused residues. The last ubiquitin moiety in many of these precursors is encoded with an extra C-terminal residue that must be removed to expose the active C-terminal Gly. In general, the recycling enzymes are thiol proteases that recognize the C-terminal domain/residue of ubiquitin. These are divided into two classes. The first is designated ubiquitin C-terminal hydrolase (UCH) and the second is designated ubiquitin-specific protease (UBP; isopeptidases) (Ciechanover, above). These enzymes have been reviewed in detail in D'Andrea, above.
UBPs contain six conserved regions. One surrounds the conserved cysteine, one surrounds the aspartic acid, one surrounds the histidine, and three additional regions of unknown function have been identified. These six domains provide a molecular signature for the UBP family. Short sequences surrounding the cysteine residue and histidine residue are highly conserved among all UBPs. Sequence comparison of several UBP family members reveals that there are various subfamilies. One subfamily, designated DUB, contains enzymes that are transcriptionally induced in response to cytokines. The UBP family contains enzymes whose members have multiple ubiquitin binding sites. Identified members of this family include DUB1, isoT, UBP3, Doa4, Tre2, and FAF (D'Andrea et al. above).
The UCH family is distinct from the UBP family. These enzymes are cysteine proteases but do not contain the six homology domains characteristic of the UBP family. Further, there is only one binding site for ubiquitin. With respect to substrate specificity, the UCH family preferentially cleaves ubiquitin from small molecules, such as peptides and amino acids. Further, the two families share little sequence homology with each other, although the UCH signature can be found in some UBPs.
The deubiquitinating enzymes can promote either degradation or stabilization of a given substrate. One of the best characterized deubiquitinating enzymes is the yeast UBP14p enzyme which has a human homolog designated isopeptidase-T. Isopeptidase-T hydrolyzes free polyubiquitin chains and stimulates degradation of polyubiquitinated protein substrates by the 26S proteasome. In vitro data suggest that the cellular role of isopeptidase-T is to dissemble unanchored polyubiquitin chains. The isopeptidase-T then sequentially degrades these polyubiquitin chains into ubiquitin monomers.
The yeast Doa4 promotes ubiquitin-mediated proteolysis of cellular substrates. The primary function appears to be the hydrolysis of isopeptide-linked ubiquitin chains from peptides that are the by-products of proteasome degradation. The function appears to be the clipping of polymeric ubiquitin from peptide degradation products. In summary, with respect to a degradation function, isopeptidases can produce free ubiquitin monomers from straight chain polyubiquitin, branched chain polyubiquitin, ubiquitin or polyubiquitin attached to substrate proteins, and ubiquitin or polyubiquitin attached to substrate remnants, such as peptides or amino acids.
Deubiquitinating enzymes that promote stabilization of substrates include the FAF protein. Results show that the FAF protein deubiquitinates and rescues a ubiquitin-conjugated target, preventing its degradation by the proteasome. Another deubiquitinating enzyme, designated PA700 isospeptidase, also prevents proteasome degradation. This enzyme has been isolated from the 19S regulatory complex. This enzyme appears to remove one ubiquitin at a time starting from the distal end of a polyubiquitin chain.
The enzymes have been associated with growth control. The mammalian oncoprotein Tre-2 is a member of the UBP superfamily. The transforming isoform of the Tre-2 oncoprotein is a truncated UPB lacking the histidine domain and lacking deubiquitinating activity. The full length Tre-2 protein has deubiquitinating activity but no transforming activity. Accordingly, it has been suggested that this protein acts as a growth suppressor within the cell.
Another UBP that regulates cellular function is designated DUB. DUB-1 was originally shown to be induced by interleukin-3 stimulation. It has been postulated that the DUB protein family is generally responsive to cytokines. It has also been shown that another family member, DUB-2, is induced by interleukin-2. Zhu et al. (1997) Journal of Biological Chemistry 272:51-57.
The enzymes may deubiquitinate cell surface growth factor receptors thereby prolonging receptor half life and amplifying growth signals. They may also deubiquitinate proteins involved in signal transduction and deubiquitinate cell cycle regulators such as cyclins or cyclin-CDK inhibitors. See D'Andrea above.
UBPs have also been linked to the chromatin regulatory process, transcriptional silencing. UBP-3 has been reported to complex with SIR-4, a trans-acting factor that is required for establishment and maintenance of silencing. Accordingly, UBP-3 may act as an inhibitor of silencing by either stabilizing an inhibitor or by removing a positive regulator.
The murine UNP protooncogene has been shown to encode a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation in NIH3T3 cells. A cDNA was cloned corresponding to the human homolog of this gene. It was shown to map to a region frequently rearranged in human tumor cells. Further, it was shown that levels of this gene are elevated in small cell tumors and adenocarcinomas of the lung, suggesting a causative role of the gene in the neoplastic process (Gray et al. (1995) Oncogene 10:2179-2183).
A novel ubiquitin-specific protease, designated UBP-43, was cloned from a leukemia fusion protein in AML1-ETO Knockin mice. This protease was shown to function in hematopoitic cell differentiation. The overexpression of this gene was shown to block cytokine-induced terminal differentiation of monocytic cells (Liu et al. (1999) Molecular and Cellular Biology 19:3029-3038).
In summary, deubiquitinating enzymes are potentially powerful targets for modulating ubiquitination. Modulation of ubiquitination can increase or decrease the proteolysis of specific proteins, particularly key proteins in cellular processes, can increase or decrease levels of general proteolysis, thus affecting the basic metabolic state, and may increase or decrease the pool of free ubiquitin monomers available for ubiquitination.
Accordingly, ubiquitin proteases are a major target for drug action and development. Thus, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown ubiquitin proteases. The present invention advances the state of the art by providing a previously unidentified human deubiquitinating enzyme.